Shunji Aoki

Reversal of the resistance to STI571 in human chronic myelogenous leukemia K562 cells.

STI571, an Abl‐specific tyrosine kinase inhibitor, selectively kills Bcr‐Abl‐containing cells in vitro and in vivo. However, some chronic myelogenous leukemia (CML) cell lines are resistant to STI571. We evaluated whether STI571 interacts with P‐glycopro‐tein (P‐gp) and multidrug resistance protein 1 (MRP1), and examined the effect of agents that reverse multidrug resistance (MDR) on the resistance to SI571 in MDR cells. STI571 inhibited the [125l]azidoagosterol A‐photolabeling of P‐gp, but not that of MRP1. K562/MDR cells that overexpress P‐gp were 3.67 times more resistant to STI571 than the parental Philadelphia‐chromosome‐positive (Ph+) CML K562 cells, and this resistance was most effectively reversed by cepharanthine among the tested reversing agents. The concentration of STI571 required to completely inhibit tyrosine phosphorylation in K562/MDR cells was about 3 times higher than that in K562 cells, and cepharanthine abolished the difference. In KB‐G2 cells that overexpress P‐gp, but not Bcr‐Abl, 2.5 μM STI571 partly reversed the resistance to vincristine (VCR), paclitaxel, etoposide (VP‐16) and actinomycin D (ACD) but not to Adriamycin (ADM) or colchicine. STI571 increased the accumulation of VCR, but not that of ADM in KB‐G2 cells. STI571 did not reverse resistance to any agent in KB/MRP cells that overexpress MRP1. These findings suggest that STI571 is a substrate for P‐gp, but is less efficiently transported by P‐gp than VCR, and STI571 is not a substrate for MRP1. Among the tested reversing agents that interact with P‐gp, cepharanthine was the most effective agent for the reversal of the resistance to STI571 in K562/ MDR cells. Furthermore, STI571 itself was a potent reversing agent for MDR in P‐gp‐expressing KB‐G2 cells.

Pyridoacridine alkaloids inducing neuronal differentiation in a neuroblastoma cell line, from marine sponge Biemna fortis.

A new and three known pyridoacridine alkaloids were isolated from the Indonesian marine sponge Biemna fortis as neuronal differentiation inducers against a murine neuroblastoma cell line, Neuro 2A. The chemical structure of the new compound, labuanine A (1), was determined by spectroscopic study and chemical conversion. These pyridoacridine alkaloids induced multipolar neuritogenesis in more than 50% of cells at 0.03-3 micro M concentration. Compound 3, which showed the strongest neuritogenic activity among them, also induced increase of acetylcholinesterase, a neuronal marker in Neuro 2A and arrested cell cycle at the G2/M phase.

Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol a on MRP1 function

We previously isolated agosterol A (AG‐A) from a marine Spongia sp. and found that it completely reversed colchicine resistance in P‐glycoprotein (Pgp)‐over‐expressing KB‐C2 cells and vincristine resistance in multidrug‐resistance protein (MRP)1‐over‐expressing CV60 cells. However, a tri‐deacetylated derivative of AG‐A (IAG‐A) showed almost no activity in reversing Pgp‐ or MRP1‐mediated drug resistance. In this study, we examined the mechanisms by which AG‐A reverses MRP1‐mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1‐over‐expressing human KB carcinoma (KB/MRP) cells. [3H]‐Leukotriene C4 (LTC4), [3H]‐2,4‐dinitrophenyl‐S‐glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [3H]‐vincristine accumulation and efflux in KB/MRP cells were measured with or without AG‐A and/or inactive IAG‐A. AG‐A reduced MRP1‐mediated [3H]‐LTC4 transport in a dose‐dependent manner, but IAG‐A did not. Inhibition by AG‐A was competitive, with a Ki value of 31 μM. AG‐A at 10 μM enhanced the accumulation of [3H]‐vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent. Likewise, ATP‐dependent efflux of [3H]‐vincristine from KB/MRP cells was enhanced compared with KB‐3‐1 cells and inhibited by AG‐A. In addition, AG‐A reduced intracellular levels of glutathione, a compound required for MRP1‐mediated transport of some anti‐cancer drugs. These findings suggest that AG‐A reverses MRP1‐mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs. In addition, the capacity of AG‐A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1.

New Rev-transport inhibitor with anti-HIV activity from Valerianae Radix.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, a HIV-1 viral regulatory protein, resulted in isolation of valtrate (1) as a new Rev-transport inhibitor from the nucleus to cytoplasm from Valerianae Radix. Valtrate (1) also inhibited the p-24 production of HIV-1 virus without showing any cytotoxicity against the host MT-4 cells.

Reversing Effect of Agosterol A, a Spongean Sterol Acetate, on Multidrug Resistance in Human Carcinoma Cells

The effect of agosterol A, a novel polyhydroxylated sterol acetate isolated from a marine sponge, on P‐glycoprotein (P‐gp)‐mediated multidrug‐resistant cells (KB‐C2) and the multidrug resistance associated protein (MRPl)‐mediated multidrug‐resistant cells (KB‐CV60) was examined. Agosterol A reversed the resistance to colchicine in KB‐C2 cells and also the resistance to vincristine in KB‐CV60 cells at 3 to 10 μM concentration. Agosterol A at 3 μM increased the vincristine concentration in both KB‐C2 cells and KB‐CV60 cells to the level in parental KB‐3‐1 cells. Agosterol A also decreased the efflux of vincristine from both KB‐C2 cells and KB‐CV60 cells to the level seen in KB‐3‐1 cells. Agosterol A inhibited the [3H]azidopine‐photolabeling of P‐gp and also inhibited the uptake of [3H]S‐(2,4‐dinitrophenyl)glutathione (DNP‐SG) in inside‐out membrane vesicles prepared from KB‐CV60 cells. We conclude that agosterol A directly inhibited drug efflux through P‐gp and/or MRP1.

Structure–activity relationship of neuritogenic spongean acetylene alcohols, lembehynes

Abstract Two new long-chain acetylene alcohols named lembehynes B ( 2 ) and C ( 3 ) were isolated from an Indonesian marine sponge of Haliclona sp. Lembehynes B ( 2 ) and C ( 3 ), which have different types of long carbon-chain parts compared with that of lembehyne A ( 1 ), also exhibited neuritogenic activity against a neuroblastoma cell line, Neuro 2A. For structure–activity relationship study of lembehynes, analogue-I ( 4 ), analogue-II ( 5 ) and analogue-III ( 6 ), which have different types of long carbon-chain parts, were synthesized from suitable fatty acids. As a result of neurite outgrowth assay for these related compounds, it was revealed that the carbon-chain length is important for neuritogenic activity, while the unsaturated bonds in the long-chain part are not. On the other hand, analogue-IV ( 7 ) with 3 S configuration showed much weaker activity than analogue-III ( 6 ) with 3 R configuration and the same type of long carbon-chain part. This indicates the importance of the stereochemistry of the hydroxyl group at C-3 in lembehynes for neuritogenic activity.

Brianthein A, a novel briarane-type diterpene reversing multidrug resistance in human carcinoma cell line, from the gorgonian Briareum excavatum

Abstract A novel briarane-type diterpene named brianthein A (1), which has been isolated from the gorgonian Briareum excavatum, reversed multidrug resistance (MDR) in human carcinoma cell lines, KB-C2, overexpressing P-glycoprotein (P-gp). The absolute stereostructure of 1 was elucidated by the detailed 2D-NMR analysis of 1 and the application of the modified Moshers method to the partially deacetylated derivative of 1. Furthermore, novel analogous compounds, briantheins B (8) and C (9), were also isolated from the same gorgonian. From the structure-activity relationship study, each of the 2, 3 and 14-acetoxyl groups and 11,12-olefin in 1 were found to be crucial for the MDR reversing activity.

Localization of the GSH-dependent photolabelling site of an agosterol A analog on human MRP1.

Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C‐terminal half of MRP1 with a photoanalog of agosterol A (azido AG‐A). In this study, we further characterized the GSH‐dependent photolabelling site of azido AG‐A on MRP1. An epitope‐inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG‐A in a GSH‐dependent manner. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti‐HA antibody suggested that the GSH‐dependent azido AG‐A photolabelling site on MRP1 resides in the region within TM14‐17 and the cytoplasmic region proximate to the C‐terminus of TM17. Arg1210 in human MRP2 that corresponds to Arg1202 in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C4 (LTC4) transport activity and conferred Vincristine resistance in LLC‐PK1 cells. In summary, this study demonstrated that the GSH‐dependent azido AG‐A photolabelling site on MRP1 resides in the region within TM14‐17 and the cytoplasmic region proximate to the C‐terminus of TM17. The charged amino acid Arg1202 proximate to TM helix 16 is of critical importance for the GSH‐dependent photolabelling of MRP1 with azido AG‐A. Arg1202 itself or the region nearby Arg1202 may be involved in azido AG‐A photolabelling.

In situ photoaffinity labeling of the target protein for lembehyne A, a neuronal differentiation inducer

A C36 linear acetylene alcohol, lembehyne A (LB‐A), induces neuronal differentiation against neuroblastoma cells morphologically and also functionally. The differentiation and cytostatic effect induced by LB‐A was specific to neuroblastoma, Neuro 2A cells. To identify the target protein for LB‐A, a radioactive photoaffinity probe, [125I]18‐(2′‐azido‐5′‐iodo‐benzoyloxy)‐LB‐18 ([125I]azido‐LB‐18), was synthesized. As a result of in situ labeling experiments against Neuro 2A cells, a protein of M r 30 kDa was photolabeled specifically. This labeling was inhibited in the presence of LB‐A or the active analogs of LB‐A, whereas the inactive analogs showed no inhibitory effect on this labeling. These results suggest that this protein of M r 30 kDa is the target protein for LB‐A and may play an important role for the neuronal differentiation in neuroblastoma, Neuro 2A cells.

Facilely accessible multidrug resistance modulator derived from sucrose.

Exploration for new MDR-modulators utilizing atractysucroses as scaffolds disclosed 2,3,4,3,4-O-pentaisovalerylsucrose (9) as a readily accessible medicinal lead. This lead was prepared from sucrose in 65% total yield for three steps. In addition, compound 9 exhibited more potent MDR modulating activity than verapamil, a representative modulator of MDR mediated by P-gp.